Transcription-driven DNA supercoiling counteracts H-NS-mediated gene silencing in bacterial chromatin.

In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is achieved dynamically, coordinated with dense and constantly changing transcriptional activity. H-NS, a major bacterial nucleoid structuring protein, is of special interest due to its interplay with RNA polymerase. H-NS:DNA nucleoprotein filaments inhibit transcription initiation by RNA polymerase. However, the discovery that genes silenced by H-NS can be activated by transcription originating from neighboring regions has suggested that elongating RNA polymerases can disassemble H-NS:DNA filaments. In this study, we present evidence that transcription-induced counter-silencing does not require transcription to reach the silenced gene; rather, it exerts its effect at a distance. Counter-silencing is suppressed by introducing a DNA gyrase binding site within the intervening segment, suggesting that the long-range effect results from transcription-driven positive DNA supercoils diffusing toward the silenced gene. We propose a model wherein H-NS:DNA complexes form in vivo on negatively supercoiled DNA, with H-NS bridging the two arms of the plectoneme. Rotational diffusion of positive supercoils generated by neighboring transcription will cause the H-NS-bound negatively-supercoiled plectoneme to "unroll" disrupting the H-NS bridges and releasing H-NS.

Widespread prevalence of a methylation-dependent switch to activate an essential DNA damage response in bacteria.

DNA methylation plays central roles in diverse cellular processes, ranging from error-correction during replication to regulation of bacterial defense mechanisms. Nevertheless, certain aberrant methylation modifications can have lethal consequences. The mechanisms by which bacteria detect and respond to such damage remain incompletely understood. Here, we discover a highly conserved but previously uncharacterized transcription factor (Cada2), which orchestrates a methylation-dependent adaptive response in Caulobacter. This response operates independently of the SOS response, governs the expression of genes crucial for direct repair, and is essential for surviving methylation-induced damage. Our molecular investigation of Cada2 reveals a cysteine methylation-dependent posttranslational modification (PTM) and mode of action distinct from its Escherichia coli counterpart, a trait conserved across all bacteria harboring a Cada2-like homolog instead. Extending across the bacterial kingdom, our findings support the notion of divergence and coevolution of adaptive response transcription factors and their corresponding sequence-specific DNA motifs. Despite this diversity, the ubiquitous prevalence of adaptive response regulators underscores the significance of a transcriptional switch, mediated by methylation PTM, in driving a specific and essential bacterial DNA damage response.

Direct observation of a crescent-shape chromosome in expanded Bacillus subtilis cells.

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genetic information. Direct visualization of chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the shape of unconfined bacterial chromosome in real-time in live Bacillus subtilis cells that are expanded in volume. We show that the chromosomes predominantly exhibit crescent shapes with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins - the key drivers of chromosomal organization - along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

Translation selectively destroys non-functional transcription complexes.

Transcription elongation stalls at lesions in the DNA template1. For the DNA lesion to be repaired, the stalled transcription elongation complex (EC) has to be removed from the damaged site2. Here we show that translation, which is coupled to transcription in bacteria, actively dislodges stalled ECs from the damaged DNA template. By contrast, paused, but otherwise elongation-competent, ECs are not dislodged by the ribosome. Instead, they are helped back into processive elongation. We also show that the ribosome slows down when approaching paused, but not stalled, ECs. Our results indicate that coupled ribosomes functionally and kinetically discriminate between paused ECs and stalled ECs, ensuring the selective destruction of only the latter. This functional discrimination is controlled by the RNA polymerase's catalytic domain, the Trigger Loop. We show that the transcription-coupled DNA repair helicase UvrD, proposed to cause backtracking of stalled ECs3, does not interfere with ribosome-mediated dislodging. By contrast, the transcription-coupled DNA repair translocase Mfd4 acts synergistically with translation, and dislodges stalled ECs that were not destroyed by the ribosome. We also show that a coupled ribosome efficiently destroys misincorporated ECs that can cause conflicts with replication5. We propose that coupling to translation is an ancient and one of the main mechanisms of clearing non-functional ECs from the genome.

Bacterial nucleoid is a riddle wrapped in a mystery inside an enigma.

Bacterial chromosome, the nucleoid, is traditionally modeled as a rosette of DNA mega-loops, organized around proteinaceous central scaffold by nucleoid-associated proteins (NAPs), and mixed with the cytoplasm by transcription and translation. Electron microscopy of fixed cells confirms dispersal of the cloud-like nucleoid within the ribosome-filled cytoplasm. Here, I discuss evidence that the nucleoid in live cells forms DNA phase separate from riboprotein phase, the "riboid." I argue that the nucleoid-riboid interphase, where DNA interacts with NAPs, transcribing RNA polymerases, nascent transcripts, and ssRNA chaperones, forms the transcription zone. An active part of phase separation, transcription zone enforces segregation of the centrally positioned information phase (the nucleoid) from the surrounding action phase (the riboid), where translation happens, protein accumulates, and metabolism occurs. I speculate that HU NAP mostly tiles up the nucleoid periphery-facilitating DNA mobility but also supporting transcription in the interphase. Besides extruding plectonemically supercoiled DNA mega-loops, condensins could compact them into solenoids of uniform rings, while HU could support rigidity and rotation of these DNA rings. The two-phase cytoplasm arrangement allows the bacterial cell to organize the central dogma activities, where (from the cell center to its periphery) DNA replicates and segregates, DNA is transcribed, nascent mRNA is handed over to ribosomes, mRNA is translated into proteins, and finally, the used mRNA is recycled into nucleotides at the inner membrane. The resulting information-action conveyor, with one activity naturally leading to the next one, explains the efficiency of prokaryotic cell design-even though its main intracellular transportation mode is free diffusion.

Antibiotic-induced ROS-mediated Fur allosterism contributes to Helicobacter pylori resistance by inhibiting arsR activation of mutS and mutY.

The increasing antibiotic resistance of Helicobacter pylori primarily driven by genetic mutations poses a significant clinical challenge. Although previous research has suggested that antibiotics could induce genetic mutations in H. pylori, the molecular mechanisms regulating the antibiotic induction remain unclear. In this study, we applied various techniques (e.g., fluorescence microscopy, flow cytometry, and multifunctional microplate reader) to discover that three different types of antibiotics could induce the intracellular generation of reactive oxygen species (ROS) in H. pylori. It is well known that ROS, a critical factor contributing to bacterial drug resistance, not only induces damage to bacterial genomic DNA but also inhibits the expression of genes associated with DNA damage repair, thereby increasing the mutation rate of bacterial genes and leading to drug resistance. However, further research is needed to explore the molecular mechanisms underlying the ROS inhibition of the expression of DNA damage repair-related genes in H. pylori. In this work, we validated that ROS could trigger an allosteric change in the iron uptake regulatory protein Fur, causing its transition from apo-Fur to holo-Fur, repressing the expression of the regulatory protein ArsR, ultimately causing the down-regulation of key DNA damage repair genes (e.g., mutS and mutY); this cascade increased the genomic DNA mutation rate in H. pylori. This study unveils a novel mechanism of antibiotic-induced resistance in H. pylori, providing crucial insights for the prevention and control of antibiotic resistance in H. pylori.

Structural basis of σ54 displacement and promoter escape in bacterial transcription.

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream of the transcription start site (TSS) and facilitates open complex formation, where double-stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to σ and upstream DNA, presumably with an enlarging transcription bubble. The release of RNAP from upstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ70 class, represented by the σ70 that regulates housekeeping genes. σ54 forms a class on its own and regulates stress response genes. Extensive studies on σ70 have revealed the molecular mechanisms of the σ70 dependent process while how σ54 transitions from initial transcription to elongation is currently unknown. Here, we present a series of cryo-electron microscopy structures of the RNAP-σ54 initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ54 and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ54 and upstream DNA, enabling the transition to elongation.

Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.

Natural transformation-specific DprA coordinate DNA double-strand break repair pathways in heavily irradiated D. radiodurans.

Deinococcus radiodurans exhibits remarkable survival under extreme conditions, including ionizing radiation, desiccation, and various DNA-damaging agents. It employs unique repair mechanisms, such as single-strand annealing (SSA) and extended synthesis-dependent strand annealing (ESDSA), to efficiently restore damaged genome. In this study, we investigate the role of the natural transformation-specific protein DprA in DNA repair pathways following acute gamma radiation exposure. Our findings demonstrate that the absence of DprA leads to rapid repair of gamma radiation-induced DNA double-strand breaks primarily occur through SSA repair pathway. Additionally, our findings suggest that the DprA protein may hinder both the SSA and ESDSA repair pathways, albeit in distinct manners. Overall, our results highlight the crucial function of DprA in the selection between SSA and ESDSA pathways for DNA repair in heavily irradiated D. radiodurans.IMPORTANCEDeinococcus radiodurans exhibits an extraordinary ability to endure and thrive in extreme environments, including exposure to radiation, desiccation, and damaging chemicals, as well as intense UV radiation. The bacterium has evolved highly efficient repair mechanisms capable of rapidly mending hundreds of DNA fragments in its genome. Our research indicates that natural transformation (NT)-specific dprA genes play a pivotal role in regulating DNA repair in response to radiation. Remarkably, we found that DprA is instrumental in selecting DNA double-strand break repair pathways, a novel function that has not been reported before. This unique regulatory mechanism highlights the indispensable role of DprA beyond its native function in NT and underscores its ubiquitous presence across various bacterial species, regardless of their NT proficiency. These findings shed new light on the resilience and adaptability of Deinococcus radiodurans, opening avenues for further exploration into its exceptional survival strategies.

Differential roles of positive and negative supercoiling in organizing the E. coli genome.

This study aims to explore whether and how positive and negative supercoiling contribute to the three-dimensional (3D) organization of the bacterial genome. We used recently published Escherichia coli GapR ChIP-seq and TopoI ChIP-seq (also called EcTopoI-seq) data, which marks positive and negative supercoiling sites, respectively, to study how supercoiling correlates with the spatial contact maps obtained from chromosome conformation capture sequencing (Hi-C and 5C). We find that supercoiled chromosomal loci have overall higher Hi-C contact frequencies than sites that are not supercoiled. Surprisingly, positive supercoiling corresponds to higher spatial contact than negative supercoiling. Additionally, positive, but not negative, supercoiling could be identified from Hi-C data with high accuracy. We further find that the majority of positive and negative supercoils coincide with highly active transcription units, with a minor group likely associated with replication and other genomic processes. Our results show that both positive and negative supercoiling enhance spatial contact, with positive supercoiling playing a larger role in bringing genomic loci closer in space. Based on our results, we propose new physical models of how the E. coli chromosome is organized by positive and negative supercoils.

The mating pilus of E. coli pED208 acts as a conduit for ssDNA during horizontal gene transfer.

IMPORTANCE: Bacteria are constantly exchanging DNA, which constitutes horizontal gene transfer. While some of these occurs by a non-specific process called natural transformation, some occurs by a specific mating between a donor and a recipient cell. In specific conjugation, the mating pilus is extended from the donor cell to make contact with the recipient cell, but whether DNA is actually transferred through this pilus or by another mechanism involving the type IV secretion system complex without the pilus has been an open question. Using Escherichia coli, we show that DNA can be transferred through this pilus between a donor and a recipient cell that has not established a tight mating junction, providing a new picture for the role of this pilus.

Phyllobacteriaceae: a family of ecologically and metabolically diverse bacteria with the potential for different applications.

The family Phyllobacteriaceae is a heterogeneous assemblage of more than 146 species of bacteria assigned to its existing 18 genera. Phylogenetic analyses have shown great phylogenetic diversity and also suggested about incorrect classification of several species that need to be reassessed for their proper phylogenetic classification. However, almost 50% of the family members belong to the genus Mesorhizobium only, of which the majority are symbiotic nitrogen fixers associated with different legumes. Other major genera are Phyllobacterium, Nitratireductor, Aquamicrobium, and Aminobacter. Nitrogen-fixing, legume nodulating members are present in Aminobacter and Phyllobacterium as well. Aquamicrobium spp. can degrade environmental pollutants, like 2,4-dichlorophenol, 4-chloro-2-methylphenol, and 4-chlorophenol. Chelativorans, Pseudaminobacter, Aquibium, and Oricola are the other genera that contain multiple species having diverse metabolic capacities, the rest being single-membered genera isolated from varied environments. In addition, heavy metal and antibiotic resistance, chemolithoautotrophy, poly-ß-hydroxybutyrate storage, cellulase production, etc., are the other notable characteristics of some of the family members. In this report, we have comprehensively reviewed each of the species of the family Phyllobacteriaceae in their eco-physiological aspects and found that the family is rich with ecologically and metabolically highly diverse bacteria having great potential for human welfare and environmental clean-up.

Single-molecule visualization of twin-supercoiled domains generated during transcription.

Transcription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake (upstream), as rotation of RNAP around the DNA axis upon tracking its helical groove gets constrained due to drag on its RNA transcript. Here, we experimentally validate this so-called twin-supercoiled-domain model with in vitro real-time visualization at the single-molecule scale. Upon binding to the promoter site on a supercoiled DNA molecule, RNAP merges all DNA supercoils into one large pinned plectoneme with RNAP residing at its apex. Transcription by RNAP in real time demonstrates that up- and downstream supercoils are generated simultaneously and in equal portions, in agreement with the twin-supercoiled-domain model. Experiments carried out in the presence of RNases A and H, revealed that an additional viscous drag of the RNA transcript is not necessary for the RNAP to induce supercoils. The latter results contrast the current consensus and simulations on the origin of the twin-supercoiled domains, pointing at an additional mechanistic cause underlying supercoil generation by RNAP in transcription.

Genotoxic stress stimulates eDNA release via explosive cell lysis and thereby promotes streamer formation of Burkholderia cenocepacia H111 cultured in a microfluidic device.

DNA is a component of biofilms, but the triggers of DNA release during biofilm formation and how DNA contributes to biofilm development are poorly investigated. One key mechanism involved in DNA release is explosive cell lysis, which is a consequence of prophage induction. In this article, the role of explosive cell lysis in biofilm formation was investigated in the opportunistic human pathogen Burkholderia cenocepacia H111 (H111). Biofilm streamers, flow-suspended biofilm filaments, were used as a biofilm model in this study, as DNA is an essential component of their matrix. H111 contains three prophages on chromosome 1 of its genome, and the involvement of each prophage in causing explosive cell lysis of the host and subsequent DNA and membrane vesicle (MV) release, as well as their contribution to streamer formation, were studied in the presence and absence of genotoxic stress. The results show that two of the three prophages of H111 encode functional lytic prophages that can be induced by genotoxic stress and their activation causes DNA and MVs release by explosive cell lysis. Furthermore, it is shown that the released DNA enables the strain to develop biofilm streamers, and streamer formation can be enhanced by genotoxic stress. Overall, this study demonstrates the involvement of prophages in streamer formation and uncovers an often-overlooked problem with the use of antibiotics that trigger the bacterial SOS response for the treatment of bacterial infections.

The bacterial replication origin BUS promotes nucleobase capture.

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).

RecN spatially and temporally controls RecA-mediated repair of DNA double-strand breaks.

RecN, a bacterial structural maintenance of chromosomes-like protein, plays an important role in maintaining genomic integrity by facilitating the repair of DNA double-strand breaks (DSBs). However, how RecN-dependent chromosome dynamics are integrated with DSB repair remains unclear. Here, we investigated the dynamics of RecN in response to DNA damage by inducing RecN from the PBAD promoter at different time points. We found that mitomycin C (MMC)-treated ΔrecN cells exhibited nucleoid fragmentation and reduced cell survival; however, when RecN was induced with arabinose in MMC-exposed ΔrecN cells, it increased a level of cell viability to similar extent as WT cells. Furthermore, in MMC-treated ΔrecN cells, arabinose-induced RecN colocalized with RecA in nucleoid gaps between fragmented nucleoids and restored normal nucleoid structures. These results suggest that the aberrant nucleoid structures observed in MMC-treated ΔrecN cells do not represent catastrophic chromosome disruption but rather an interruption of the RecA-mediated process. Thus, RecN can resume DSB repair by stimulating RecA-mediated homologous recombination, even when chromosome integrity is compromised. Our data demonstrate that RecA-mediated presynapsis and synapsis are spatiotemporally separable, wherein RecN is involved in facilitating both processes presumably by orchestrating the dynamics of both RecA and chromosomes, highlighting the essential role of RecN in the repair of DSBs.

RNA is a key component of extracellular DNA networks in Pseudomonas aeruginosa biofilms.

The extracellular matrix of bacterial biofilms consists of diverse components including polysaccharides, proteins and DNA. Extracellular RNA (eRNA) can also be present, contributing to the structural integrity of biofilms. However, technical difficulties related to the low stability of RNA make it difficult to understand the precise roles of eRNA in biofilms. Here, we show that eRNA associates with extracellular DNA (eDNA) to form matrix fibres in Pseudomonas aeruginosa biofilms, and the eRNA is enriched in certain bacterial RNA transcripts. Degradation of eRNA associated with eDNA led to a loss of eDNA fibres and biofilm viscoelasticity. Compared with planktonic and biofilm cells, the biofilm matrix was enriched in specific mRNA transcripts, including lasB (encoding elastase). The mRNA transcripts colocalised with eDNA fibres in the biofilm matrix, as shown by single molecule inexpensive FISH microscopy (smiFISH). The lasB mRNA was also observed in eDNA fibres in a clinical sputum sample positive for P. aeruginosa. Thus, our results indicate that the interaction of specific mRNAs with eDNA facilitates the formation of viscoelastic networks in the matrix of Pseudomonas aeruginosa biofilms.

Dynamic ParB-DNA interactions initiate and maintain a partition condensate for bacterial chromosome segregation.

In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, using single-molecule fluorescence microscopy and AFM imaging to show that transient ParB-ParB bridges are essential for forming DNA condensates. Molecular Dynamics simulations confirm that condensation occurs abruptly at a critical concentration of ParB and show that multimerization is a prerequisite for forming the partition complex. Magnetic tweezer force spectroscopy on mutant ParB proteins demonstrates that CTP hydrolysis at the N-terminal domain is essential for DNA condensation. Finally, we show that transcribing RNA polymerases can steadily traverse the ParB-DNA partition complex. These findings uncover how ParB forms a stable yet dynamic partition complex for chromosome segregation that induces DNA condensation and segregation while enabling replication and transcription.

A method for assaying DNA flexibility.

The transcription, replication, packaging, and repair of genetic information ubiquitously involves DNA:protein interactions and other biological processes that require local mechanical distortions of DNA. The energetics of such DNA-deforming processes are thus dependent on the local mechanical properties of DNA such as bendability or torsional rigidity. Such properties, in turn, depend on sequence, making it possible for sequence to regulate diverse biological processes by controlling the local mechanical properties of DNA. A deeper understanding of how such a "mechanical code" can encode broad regulatory information has historically been hampered by the absence of technology to measure in high throughput how local DNA mechanics varies with sequence along large regions of the genome. This was overcome in a recently developed technique called loop-seq. Here we describe a variant of the loop-seq protocol, that permits making rapid flexibility measurements in low-throughput, without the need for next-generation sequencing. We use our method to validate a previous prediction about how the binding site for the bacterial transcription factor Integration Host Factor (IHF) might serve as a rigid roadblock, preventing efficient enhancer-promoter contacts in IHF site containing promoters in E. coli, which can be relieved by IHF binding.

Proteomic survey of the DNA damage response in Caulobacter crescentus.

The bacterial DNA damage response is a critical, coordinated response to endogenous and exogenous sources of DNA damage. Response dynamics are dependent on coordinated synthesis and loss of relevant proteins. While much is known about its global transcriptional control, changes in protein abundance that occur upon DNA damage are less well characterized at the system level. Here, we perform a proteome-wide survey of the DNA damage response in Caulobacter crescentus. We find that while most protein abundance changes upon DNA damage are readily explained by changes in transcription, there are exceptions. The survey also allowed us to identify the novel DNA damage response factor, YaaA, which has been overlooked by previously published, transcription-focused studies. A similar survey in a ∆lon strain was performed to explore lon's role in DNA damage survival. The ∆lon strain had a smaller dynamic range of protein abundance changes in general upon DNA damage compared to the wild-type strain. This system-wide change to the dynamics of the response may explain this strain's sensitivity to DNA damage. Our proteome survey of the DNA damage response provides additional insight into the complex regulation of stress response and nominates a novel response factor that was overlooked in prior studies. IMPORTANCE The DNA damage response helps bacteria to react to and potentially survive DNA damage. The mutagenesis induced during this stress response contributes to the development of antibiotic resistance. Understanding how bacteria coordinate their response to DNA damage could help us to combat this growing threat to human health. While the transcriptional regulation of the bacterial DNA damage response has been characterized, this study is the first to our knowledge to assess the proteomic response to DNA damage in Caulobacter.